Made available in DSpace on 2015-07-22T20:50:18Z (GMT). No. of bitstreams: 0
Previous issue date: 2009-08-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) / World Health Organization (WHO) / Howard Hughes Medical Institute / Proteinas de adesao celular sao essenciais para a invasao celular do hospedeiro mamifero pelo parasita Trypanosoma cruzi. Neste trabalho, nos mostramos que membros de gDispersed Gene Family-1 h, originalmente descrita como uma familia de sequencias nucleares repetitivas presente em varios cromossomos e compondo a terceira maior familia genica especifica deste parasita, contem caracteristicas de adesinas, incluindo quatro segmentos com similaridade significante a sequencia da subunidade ƒÀ7 de integrina humana. Ensaios de biotinilacao da superficie de parasitas e citometria de fluxo com anticorpos anti-DGF-1 indicaram que os membros dessa familia genica sao expressos na superficie das formas tripomastigotas. A genealogia de DGF-1, inferida por algoritmos de rede a partir de dados do Projeto Genoma da linhagem CL Brener de T. cruzi, sugere que essa familia genica pode ser dividida em pelo menos tres grupos com diferentes padroes de distribuicao de seus dominios funcionais. Analisando o perfil de uso dos codons, observou-se que as copias expressas apresentavam um uso preferencial de codons, favorecendo as bases GC, ao passo que copias nao-expressas, incluindo alguns pseudogenes, apresentavam uma distribuicao homogenea no uso de codons. A entropia informacional de Shannon foi utilizada como uma medida da variabilidade de sequencia e revelou que quatro segmentos de alta entropia coincidem com modulos funcionais putativos das proteinas preditas. Contradizendo a ideia de que alta variabilidade esta associada a selecao positiva, os resultados de dois testes distintos de selecao mostraram que posicoes altamente variaveis nao estao necessariamente sob selecao positiva. Nossa hipotese e de que membros de DGF-1 estao associados com a habilidade do T. cruzi se ligar a proteinas da matriz extracelular, como fibronectina e laminina, e especulamos sobre os mecanismos que gerariam diversidade nessas moleculas na ausencia de selecao. / Surface adhesion proteins are essential for Trypanosoma cruzi invasion of mammalian cells. Here we show that Dispersed Gene Family-1 (DGF-1) members, previously identified as nuclear repeated sequences present in several chromosomes and comprising the third largest T. cruzi-specific gene family, have conserved adhesin motifs including four segments with significant similarity to human beta 7 integrin. Flow cytometry and biotinylation assays with anti-DGF-1 antibodies indicated that, as expected, DGF-1 members are expressed on the trypomastigote surface. The DGF-1 genealogy, inferred using T. cruzi Genome Project data and network phylogeny algorithms, suggests that this gene family is separated in at least three groups with differential distribution of functional domains. To identify which members of this gene family are expressed we used a combined approach of RT-PCR and codon usage profiles, showing that expressed members have a very biased codon usage favoring GC whereas non-expressed members have a homogeneous distribution. Shannon information entropy was used to measure sequence variability and revealed four major high entropy segments in the extracellular domain of DGF-1 overlapping with important putative functional modules of the predicted proteins. Testing for natural selection, however, indicated that these high entropy segments were not under positive selection, which contradicts the notion that positive selection is the cause of high variability in specific domains of a protein relative to other less variable regions in the same molecule. We hypothesize that members of the DGF-1 family are associated with the ability of T. cruzi to bind extracellular matrix proteins, such as fibronectin and laminin, and speculate on mechanisms that would be generating the localized diversity in these molecules in the absence of selection. / FAPESP: 03/05317-0 / TEDE / BV UNIFESP: Teses e dissertações
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.unifesp.br:11600/9695 |
| Date | 26 August 2009 |
| Creators | Kawashita, Silvia Yukie [UNIFESP] |
| Contributors | Universidade Federal de São Paulo (UNIFESP), Briones, Marcelo Ribeiro da Silva [UNIFESP] |
| Publisher | Universidade Federal de São Paulo (UNIFESP) |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | 131 f. |
| Source | reponame:Repositório Institucional da UNIFESP, instname:Universidade Federal de São Paulo, instacron:UNIFESP |
| Rights | info:eu-repo/semantics/openAccess |
Page generated in 0.0026 seconds